In the fields of histology, pathology, and cell biology, fixation is a chemical process by which biological samples are preserved from decay. Fixation terminates any ongoing biochemical reactions, and may also increase the mechanical strength or stability of the treated samples. The purpose of fixation is to preserve a sample of biological material as close to its natural state as possible. Fixed samples are used for examination or analysis.
Immersion is a fixation technique in which the sample is immersed in fixative of volume at a minimum of 20 times greater than the volume of the tissue to be fixed. The fixative diffuses through the tissue in order to fix, so tissue size and density, as well as the type of fixative must be taken into account. Using a larger sample means it will take longer for the fixative to reach the deeper tissue.
Fixative agents can be classified as crosslinking or precipitative fixatives. Crosslinking fixatives act by creating covalent chemical bonds between proteins in tissue. This anchors soluble proteins to the cytoskeleton, and lends additional rigidity to the tissue. Precipitating, or denaturing, fixatives act by reducing the solubility of protein molecules and, often, by disrupting the hydrophobic interactions which give many proteins their tertiary structure.
One commonly used fixative in histology is the crosslinking fixative formaldehyde, which is often sold as a saturated aqueous solution under the name formalin. Formaldehyde is thought to interact primarily with the residues of the basic amino acid lysine.
Another popular aldehyde for fixation is glutaraldehyde, which is believed to operate by a similar mechanism to formaldehyde.
Formaldehyde preserves or fixes tissue or cells by cross-linking primary amino groups in proteins or nucleic acids through a —CH2— linkage, i.e., a methylene bridge. Because formaldehyde is highly reactive, excessive formaldehyde in the sample or media interferes with any sample processing or analysis that involves functional proteins (such as enzymes or antibodies), nucleic acid probes, resins, or any other functional reagents with amino groups by cross-linking to these amino groups with subsequent reagent deactivation. Moreover, since the cross-links can be reversed by heat, any excessive formaldehyde in the media will eventually form cross links again, preventing cross-link reversal from being effective.
Oxidizing fixatives can react with various side chains of proteins and other biomolecules, allowing the formation of crosslinks which stabilize tissue structure. Osmium tetroxide is often used as a secondary fixative when samples are prepared for electron microscopy. Potassium dichromate, chromic acid, and potassium permanganate are also used in specific histological preparations. Two common precipitating fixatives are ethanol and methanol. Acetone is also used.
Acetic acid is a denaturant that is sometimes used in combination with other precipitating fixatives. Alcohols, by themselves, are known to cause shrinkage of tissue during fixation while acetic acid alone is associated with tissue swelling; combining the two may result in better preservation of tissue morphology. Other fixative agents include picric acid and mercuric chloride.
One of the problems with fixing biological samples is that the nucleic acids and proteins in the samples may be irreversibly bound to the fixative agent(s). Even if the nucleic acids and proteins are not irreversibly bound to the fixative agent(s), removal of excess fixative agents from the samples may be important for reliable recovery and analysis of nucleic acids and proteins. Additionally, fixative agents may interfere with the use of the isolated protein or nucleic acid in downstream biochemical analyses, such as PCR.